CN101565690B - Enrofloxacin monoclonal antibody and application - Google Patents

Abstract

The invention relates to an enrofloxacin monoclonal antibody and application, relates to hybridoma strains thereof, and belongs to the technical field of immunochemistry. The enrofloxacin monoclonal antibody is generated by mouse hybridoma strains 6A4 and 8E6. The preparation method comprises the following steps that: enrofloxacin and carrier proteins BSA, HAS and OVA are coupled by a carbodiimidemethod to synthesize artificial immunogens EnR-BSA, EnR-HSA and coatingen EnR-OVA; a Balb/c mouse is immunized by the synthesized artificial immunogens EnR-BSA and EnR-HSA; a spleen cell of the immunized mouse is extracted to be fused with a SP2/O myeloma cell and coated by the coatingen EnR-OVA; indirect ELISA method and indirect competition ELISA method are established to screen the hybridoma strains which can stably secrete specific antibody; the obtained cell strain immunized Balb/c mouse is used to prepare ascites; the ascites is purified by a caprylic acid-ammonium method and an ion exchange method; and valences of antibodies of two purified cell strains reach over 1.024*10<-6> and 1.28*10<-5>. The monoclonal antibody has strong specificity, can be applied to preparation of enrofloxacin residue inspection kit and aerosol test strip, and can sensibly and quickly inspect the enrofloxacin residue.

Description

Enrofloxacin monoclonal antibody and application

Technical field

The present invention relates to monoclonal antibody specific of Enrofloxacin and its production and application, also relate to its hybridoma cell strain, belong to the immunochemical technique field.

Background technology

Enrofloxacin (enrofloxacin) is the fluoroquinolones of first livestock and poultry special use, and has a broad antifungal spectrum, anti-microbial activity are strong, the advantage such as wide that easily absorbs, distributes in the body is widely used in veterinary clinic prevention and treatment because of it has.Along with the widespread use of this medicine in food source property animal, also increasing by its toxic side effect that causes such as neurotoxic, renal dysfunction, anaphylaxis, cub sacroiliitis etc.Owing to the residual discovery of inducing human disease bacterium produce resistance problem of this medicine in animal food, further deepened the understanding of people to this medicine.In order to reduce the harm of this medicine to the mankind, the U.S. forbids Enrofloxacin is used for food animal, and Japanese MHLW from May, 2007 with fish, seashell products in residual the limiting the quantity of of Enrofloxacin be adjusted into 0.01ppm by 0.1ppm.Therefore, it is most important to carry out the research of its method for detecting residue.In order to monitor Enrofloxacin residual in animal food, Food and Argriculture OrganizationFAO (FAO)/The World Health Organization (WHO) and the foodstuff additive joint specialist council (JCEFA) formulated Enrofloxacin the maximum residue limit(MRL) of ox, pig, fowl (maximum residue limit, MRL).U.S. FDA regulation Enrofloxacin is forbidden in food animal, and European Union (EC) regulation Enrofloxacin maximum residue limit(MRL) (MRL) in the liver of ox, pig, poultry, kidney, muscle is 30 μ g/kg; The Enrofloxacin MRL that The World Health Organization (WHO) is recommended is 40 μ g/kg.The MRL of regulation Enrofloxacin in muscle, milk of China Ministry of Agriculture must not surpass 100 μ g/kg.Therefore residue detection and the supervision in animal food seems particularly outstanding to this medicine in reinforcement.Yet can not induce body to produce antibody as haptenic Enrofloxacin itself, its complete antigen with carrier protein couplet acquisition synthetic just must be had antigenicity.And the screening of positive colony all directly affects the preparation effect of antibody after the coupling effect of small molecules antigen and carrier proteins and the cytogamy, is the key of Antibody Preparation.In order to set up Enrofloxacin enzyme immunoassay method and gold test strip, need the monoclonal antibody of preparation high specificity, the anti-Enrofloxacin of mass producible.

The applicant retrieves and finds Scientia Agricultura Sinica (2004,37 (7): 1 060-1 064) preparation and the evaluation thereof of enrofloxacin monoclonal antibody have been introduced in one piece of document, mainly be to have synthesized 2 kinds of Enrofloxacin (enrofloxacin with carbodlimide method, ENFX) artificial antigen ENFX-BSA and ENFX-OVA, methods such as amino acid composition with UV scanning and analysis carrier proteins identify that to artificial antigen the binding ratio of ENFX and BSA is about 48: 1.Use the ENFX-BSA immune balb/c mice, adopt microclone technology finishing screen to select 2 strain specific secretion cell 2C5 and 5D5 and obtain ascites, ascites is tired and is respectively 1: 3200 and 1: 6 400 behind the purifying.2C5 and 5D5 all belong to IgG2a type antibody.This monoclonal antibody has very high selectivity to fluoroquinolones, be respectively 110.84%, 27.40%, 71.05% and 37.41% with the cross reacting rate of Ciprofloxacin, marbofloxacin, sarafloxacin, danofloxacin, cross reacting rate is than higher, and specificity is not strong.

Summary of the invention

The objective of the invention is: at the shortcoming of above prior art existence, proposition and Ciprofloxacin and sarafloxacin no cross reaction, and have specific, mass producible enrofloxacin monoclonal antibody.

Another object of the present invention provides the hybridoma cell strain of the anti-enrofloxacin monoclonal antibody of this specificity of secretion.

The objective of the invention is to be achieved through the following technical solutions:

The present invention produces the hybridoma cell strain 6A4 or the 8E6 of enrofloxacin monoclonal antibody, 6A4 is that mouse hybridoma cell is CGMCC No.3016, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 9th, 2009, preserving number is: CGMCCNo.3016; 8E6 is that mouse hybridoma cell is CGMCC No.3017, is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 9th, 2009, and preserving number is: CGMCC No.3017.

By mouse hybridoma cell strain 6A4 or mouse hybridoma cell strain 8E6 excretory monoclonal antibody.

Mouse hybridoma cell strain 6A4 or mouse hybridoma cell strain 8E6 excretory monoclonal antibody the preparation method, may further comprise the steps:

A. prepare immunogen and coating antigen: adopt carbodlimide method with carrier proteins and Enrofloxacin coupling, synthetic immunogen and coating antigen;

B. animal immune injection: as immune animal, abdominal injection immunogen and equivalent Freund's complete adjuvant mixed solution carry out the immunization first time with mouse; Carry out the immunization second time after 3 weeks; Carry out immunization for the third time after 2 weeks at interval again;

C. screen animal immune serum:, mouse is carried out the booster immunization injection with enzyme-linked immunosorbent assay and competitive enzyme-linked immune absorption method screening immune mouse serum;

D. prepare hybridoma: splenocyte of the mouse of the booster immunization of learning from else's experience and myeloma cell carry out cytogamy, through the cell strain of monoclonal antibody 6A4 or the 8E6 of 3 anti-Enrofloxacin of subclone acquisition stably excreting;

E. prepare and monoclonal antibody purification: mouse is carried out immunity with sterilization paraffin, difference described cell strain of monoclonal antibody 6A4 of abdominal injection or 8E6 after 7 days, gathered ascites, and to the ascites purifying, promptly got enrofloxacin monoclonal antibody in 7-10 days with sad-ammonium sulfate salting-out process.

Wherein, carrier proteins described in the step a is at least a of bovine serum albumin, human serum albumin and oralbumin, and corresponding immunogen is ENR-BSA, ENR-HAS, and coating antigen is ENR-OVA; In the described carbodlimide method, the reaction times of EDC and NHS activation Enrofloxacin is 2 hours, Enrofloxacin and carrier proteins reaction 12 hours.

Each time immunization dosage among the described step b is 50-100 μ g/.

In the described steps d, before carrying out cytogamy, immune mouse serum is utilized the indirect ELISA method detection of tiring, utilize the indirect competitive ELISA method to detect the inhibition effect of serum simultaneously, merge with the mice spleen cell after the detection to Enrofloxacin.

The injection volume that among the described step e cell strain of monoclonal antibody is carried out in the immune mouse abdominal cavity is 1-5 * 10 ⁶Individual/only.

Identify that by hypotype the hypotype that draws monoclonal antibody of the present invention is the IgG1 type.

A further object of the present invention provides the application of this monoclonal antibody in preparation detection Enrofloxacin reagent.It mainly is to be applied to prepare enrofloxacin residual detection kit and Radioactive colloidal gold strip paper.

The invention has the beneficial effects as follows: the enrofloxacin monoclonal antibody that produces high specificity by two strain mouse hybridoma cell strain 6A4 and 8E6, low with other fluoroquinolones cross reacting rate, can mass production, can be used for preparing the enzyme-linked immunosorbent assay test kit and the colloidal gold strip that detect Enrofloxacin, reach and detect milk fast and delicately, the effect of the enrofloxacin medicament residue in muscle and the fishery products.

Description of drawings

The present invention is further illustrated below in conjunction with accompanying drawing.

Fig. 1: ENR-HSA immunogen mass spectrometric detection figure.

Fig. 2: ENR-BSA immunogen mass spectrometric detection figure.

Fig. 3: ENR-OVA coating antigen mass spectrometric detection figure.

Fig. 4: the hypotype of two monoclonal antibody 8E6 ' and 6A4 ' is identified figure.

Fig. 5: the response curve of monoclonal antibody 8E6 ' of the present invention and Enrofloxacin standard substance.

Fig. 6: the response curve of monoclonal antibody 6A4 ' of the present invention and Enrofloxacin standard substance.

Embodiment

Embodiment one

Reagent and material are prepared

Enrofloxacin standard substance (China Veterinery Drug Inspection Office), bovine serum albumin (BSA) (Amresco, 0332), human serum albumin (HSA) (Sigma, A9511), oralbumin (OVA) (Sigma, A5503), and carbodiimide hydrochloride (EDC) (Sigma, E6383), N-hydroxy-succinamide (NHS) (Sigma, 130672), N, dinethylformamide (DMF) (Sigma, D4551), Freund's complete adjuvant (Sigma, F5881), Freund's incomplete adjuvant (Sigma, F5506), tetramethyl benzidine (TMB) (Amresco, 0759), HAT (Sigma, H0262) and HT (Sigma, H0137), sodium-chlor (Amresco, 0241), Repone K (Amresco, 0395), and potassium primary phosphate (Sigma, P9791), Sodium phosphate dibasic (Sigma, 71639), and sheep anti-mouse igg-HRP (Jackson, 115-035-044), dimethyl sulfoxide (DMSO) (DMSO) (Applichem, 0231), and Macrogol 4000 (PEG4000) (Sigma, P7306), DMEM high glucose medium (Gibco, 11995), and foetal calf serum (Gibco, C2027050).

Laboratory animal and cell: Balb/c mouse (6-8 week age, female), available from Beijing Vital River Experimental Animals Technology Co., Ltd., SP2/0 (murine myeloma cell) is infected with immune Research center professor Tang Jie by biophysics institute of the Chinese Academy of Sciences and is so kind as to give.

Experimental procedure of the present invention is as follows:

A. prepare immunogen ENR-BSA, ENR-HSA and coating antigen ENR-OVA:

Artificial antigen of the present invention is adopted carbodlimide method ^[1]Preparation enrofloxacin and carrier proteins mixture, and slightly do improvement:

(1) DMF that gets 100 μ L dissolves 2.4mg respectively, 2.5mg, and the Enrofloxacin of 1.6mg adds among the PBS (pH8.0) of 100 μ L mixing with the Enrofloxacin after the dissolving;

(2) the earthquake state slowly adds the EDC of 150 μ L 0.3M down, continues to add the NHS of 150 μ L 0.2M, room temperature reaction 2h;

(3) reaction product is contained 10mg BSA slow the adding, in PBS (pH8.0) solution of the 500 μ L of HSA and OVA, 4 ℃ of lucifuges were reacted 12 hours;

(4) above-mentioned reaction product is packed into dialysis tubing (molecular weight cut-off 3500) is dialysed, and changes liquid once in 2 hours, changes liquid altogether 3 times, leaves heart 30min with 13000, collects supernatant, packing be stored in-20 ℃ standby.

(5) to the immunogen ENR-HSA after the dialysis, ENR-BSA and coating antigen ENR-OVA carry out mass spectrometric detection respectively, the results are shown in Figure 1, Fig. 2, Fig. 3.Fig. 1 shows that the coupling ratio of conjugate ENR-HSA and carrier proteins is 9: 1; Fig. 2 shows that the coupling ratio of conjugate ENR-BSA and carrier proteins BSA is 5: 1; Fig. 3 shows that the coupling ratio of conjugate ENR-OVA and carrier proteins OVA is 9: 1, can judge the coupling success from the diagram result.

B. animal immune:

Adopt the Balb/c mouse as immune animal, immunogen is ENR-HSA or ENR-BSA, and immunizing dose is 50-100 μ g/, and for the first time with immunogen and equivalent Freund's complete adjuvant mixing and emulsifying, abdominal injection, dosage are 100 μ g/0.2ml/; Carry out the immunization second time with immunogen and equivalent Freund's incomplete adjuvant mixing and emulsifying after 3 weeks, dosage is 50 μ g/0.2ml/, after 2 weeks, carries out immunization for the third time with Freund emulsive antigen at interval, and dosage is the same.

C. screen animal immune serum:

Above immune mouse was surveyed serum titer at immune back 7-10 days for the third time with the ELISA method, utilized the indirect competitive ELISA method simultaneously ^[2]Detect the inhibition effect of serum to Enrofloxacin.Choose the high mouse of serum titer and carry out booster immunization, immunity was got spleen after three days.

D. prepare hybridoma:

Get the splenocyte of above-mentioned immune Balb/c mouse, the PEG4000 with 50% makes fusogen, and immune spleen cell and SP2/0 myeloma cell are carried out cytogamy in 5: 1 ratio.Adopt indirect elisa method to detect cell conditioned medium, filter out positive colony, positive colony is further detected with the indirect competitive ELISA method, still positive person utilizes limiting dilution assay to carry out subclone, after 3 subclones, detection has the cells and supernatant in mono-clonal growth hole, and positive rate reaches 100%, obtains the hybridoma cell strain 6A4 and the 8E6 of stably excreting monoclonal antibody;

The step of setting up indirect elisa method in this step is as follows:

Best antigen coated dilution selection, adopt the square formation method to determine coating antigen concentration:

(1) bag quilt: the carbonate buffer solution with 0.05mol/L, PH9.6 is diluted to coating antigen in a series of concentration adding enzyme plates, 100 μ L/ holes, and 4 ℃ of bags are spent the night;

(2) washing: get rid of clean coating buffer, washings is the PBS that contains the 0.01mol/L PH7.4 of 1 ‰ tweens, with washing machine-wash plate 3 times of plate, and pats dry on thieving paper;

(3) sealing: every hole adds the 0.5%BSA confining liquid of 200 μ L, hatches 1h for 37 ℃;

(4) washing is the same;

(5) one is anti-: add the antibody of series concentration, 1h is hatched for 37 ℃ in 100 μ L/ holes;

(6) washing is the same;

(7) ELIAS secondary antibody: add the sheep anti mouse-HRP of dilution in 1: 10000,1h is hatched for 37 ℃ in 100 μ L/ holes, washs the same;

(8) colour developing: every hole adds the TMB colour developing liquid of 100 μ L, 20-25 ℃ of colour developing 10min;

(9) termination reaction: the H that adds 2M ₂SO ₄The stop buffer termination reaction, 50 μ L/ holes, and on microplate reader, read OD ₄₅₀Value.

Criterion:, determine that the diluted degree of the best bag of coating antigen is 1: 28000 (concentration is 0.1 μ g/ml) according to positive serum OD value and P/N 〉=2.1.Concrete outcome sees Table 1

Table 1 antigen, antibody best effort concentration

The step of setting up the indirect competitive ELISA method in this step is as follows:

(1) bag quilt: the carbonate buffer solution with 0.05mol/L, PH9.6 is 0.1ug/ml with the coating antigen dilution, adds in the enzyme plate, and 100 μ L/ holes, 4 ℃ of bags are spent the night;

(2) washing: get rid of clean coating buffer, washings is the PBS that contains the 0.01mol/L PH7.4 of 1 ‰ tweens, with washing machine-wash plate 3 times of plate, and pats dry on thieving paper;

(3) sealing: every hole adds the 0.5%BSA confining liquid of 200 μ L, hatches 1h for 37 ℃;

(4) washing is the same;

(5) application of sample: earlier will be in the enzyme plate hole, adjacent two hole in the adding diluent, 50 μ L/ holes, the Enrofloxacin standard substance of adding proper concn in the adjacent hole, two holes therewith, 50 μ L/ holes add the good antibody of dilution, 50 μ L/ holes at last, enzyme plate makes to shake mixing slightly, hatches 1h for 37 ℃;

(6) washing is the same;

(7) ELIAS secondary antibody: add the sheep anti mouse-HRP of dilution in 1: 10000,1h is hatched for 37 ℃ in 100 μ L/ holes, washs the same;

(8) colour developing: every hole adds the TMB colour developing liquid of 100 μ L, 20-25 ℃ of colour developing 10min;

(9) termination reaction: the H that adds 2mol/L ₂SO ₄The stop buffer termination reaction, 50 μ L/ holes, and on microplate reader, read OD ₄₅₀Value.

Criterion: at first, from naked eyes as can be seen, suppress Kong Yuwei and suppress the hole change in color,, illustrated that specific antibody produces, do not produce otherwise then there is specific antibody if it is more shallow or colourless to suppress the hole color.Secondly, can judge, suppress hole OD value, illustrate that specific antibody produces less than not suppressing hole OD value according to the OD value.Employing has the immune mouse spleen cell that suppresses effect serum to carry out cytogamy, and filters out the hybridoma cell strain of energy secreting specificity antibody.

The power of antibodies specific can be judged according to the size of competition inhibiting rate.

Competition inhibiting rate=1-B/B0 (B adds the inhibition hole OD value of competing thing, and B0 is not for adding the positive control hole OD value of competing thing).

E. prepare and monoclonal antibody purification titration:

At first prepare and identify monoclonal antibody: take out frozen pipe during cell recovery, melt in 37 ℃ of water-baths immediately, move into enlarged culturing in the culture dish afterwards, substratum is the DMEM substratum that contains 10%FBS.Wherein, in the frozen pipe frozen be logarithmic phase can the stably excreting monoclonal antibody hybridoma cell strain 6A4 and 8E6.

With 8 Balb/c mouse of sterilization paraffin immunity, 0.5ml/ only is divided into two groups with mouse, and 4 every group, respectively to every group of mouse peritoneal injection hybridoma 6A4 and 8E6, injected dose is 1-5 * 10 after 7 days ⁶Individual/as only, to gather ascites, and obtained monoclonal antibody, called after 6A4 ' and 8E6 ' in 7-10 days.Employing is carried out the hypotype evaluation available from the mouse source monoclonal antibody hypotype identification kit (Mouse Monoclonal Antibody SubtypeIdentification Kit) of Pierce company to the resulting monoclonal antibody of the present invention, its hypotype qualification result shows, monoclonal antibody 6A4 ' and 8E6 ' are the IgG1 hypotype, different with the IgG2a hypotype of existing bibliographical information, the results are shown in Table 2 and Fig. 4.

Antibody subtype	8E6?clone	6A4?clone
			IgA	0.223	0.228
IgG3	0.200	0.200
			IgM	0.190	0.199
IgG1	1.444	1.513
			IgG2a	0.296	0.294
IgG2b	0.197	0.199
			The κ chain	0.929	1.045
The λ chain	0.244	0.216
			blank?control	0.204	0.240
negative	0.220	0.220

Table 2 antibody subtype qualification result

Monoclonal antibody purification (hereinafter to be referred as monoclonal antibody) then: with sad-ammonium sulfate salting-out process above-mentioned ascites is handled, concrete steps are as follows:

(1) gets the ascites of 1 times of volume, add the NaAC-HAC damping fluid of the 0.06mol/L PH4.8 of 4 times of volumes, gently mixing;

(2) add 30% sad amount by every ml ascites, add good after, room temperature is shaken 30min on shaking table.4 ℃ leave standstill 3h afterwards;

(3) 4 ℃ at 13000rpm, and centrifugal 30min gets supernatant, transfers PH to 7.4 with Na0H;

(4) in supernatant, add isopyknic saturated ammonium sulphate solution, make its final concentration be 50%, 4 ℃ and leave standstill 1h.4 ℃ of 13000rpm afterwards, centrifugal 10min;

(5) abandon supernatant, with the PBS suspension precipitation of an amount of 0.01mol PH7.2;

(6) change the monoclonal antibody suspension over to the 12h that dialyses in the dialysis tubing, the centre is changed dialyzate 3 times;

(7) monoclonal antibody after the dialysis is made of ion exchange method and is further purified, used ion exchange column is HiTrap Q HP 1mL, sample-loading buffer is 20mM Tris-HCL, pH8.6, elution buffer is 20mM Tris-HCL, 1MNaCL, pH8.6, when reaching 30%, salt concn begins to collect monoclonal antibody, the 1ml/ pipe.

(8) monoclonal antibody behind the purifying carries out its purity of SDS-PAGE electrophoresis detection.Utilize ultraviolet spectrophotometer to measure its concentration, monoclonal antibody is adjusted into 1mg/ml, survey it then and tire, packing ,-80 ℃ of preservations according to measurement result;

(9) the monoclonal antibody mensuration of tiring: with the coating antigen ENR-OVA bag of 0.1 μ g/ml by elisa plate, 6A4 ' the monoclonal antibody of purifying was carried out 1: 2000,1: 4000,1: 8000,1: 16000,1: 32000,1: 64000, dilution in 1: 128000, add in the enzyme plate hole, the reaction back adds the sheep anti-mouse igg of HRP mark, with the TMB colour developing, the results are shown in Table 3 at last; The mensuration that 8E6 ' monoclonal antibody is tired, the mensuration that the same 6A4 ' monoclonal antibody of method is tired, the monoclonal antibody extent of dilution was respectively 1: 8000,1: 16000,1: 32000,1: 64000,1: 128000,1: 256000,1: 512000, the results are shown in Table 3 at 1: 1024000.

The titration result of table 3 6A4 ' and 8E6 '

Positive criterion: P/N 〉=2.1

6A4 ' antibody test result: when antibody purification concentration was 1mg/ml, tiring to reach 1.28 * 10 ^-5More than;

8E6 ' antibody test result: when antibody purification concentration was 1mg/ml, tiring to reach 1.024 * 10 ^-6More than.

(10) specificity of detection purified monoclonal antibody: adopt the indirect competitive ELISA method of setting up in the steps d to carry out, be coated with in the antigenic elisa plate, 6A4 ' (0.05 μ g/ml) and 8E6 ' (0.05 μ g/ml) antibody behind the adding purifying, the Enrofloxacin standard substance that add different concns simultaneously, be added with the Enrofloxacin concentration that adds in the hole of 8E6 ' antibody and be respectively 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL, 0.313ng/mL, 0ng/mL, be added with the Enrofloxacin concentration that adds in the hole of 6A4 ' antibody and be respectively 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL, 3.125ng/mL, 0ng/mL, replication 3 times, the result sees Table 4 respectively, table 5.Natural logarithm with drug level is an X-coordinate, is that ordinate zou is drawn the mark song with the B/B0 value, the results are shown in Figure 5, Fig. 6.The IC50 value of calculating 6A4 ' and 8E6 ' monoclonal antibody according to testing data is respectively 14ng/ml and 2.3ng/ml.

From the OD value of typical curve and IC50 value as can be seen, when 6A4 ' monoclonal antibody is 3.125ng/ml in ENR concentration, still has the good restraining effect; And 8E6 ' monoclonal antibody is when ENR concentration is 0.313ng/ml, and it is obvious to suppress effect, and the two strain monoclonal antibodies that screen among this explanation the present invention have very high specificity and susceptibility.

The inhibition effect of table 4 monoclonal antibody 8E6 '

The inhibition effect of table 5 monoclonal antibody 6A4 '

(11) mensuration of monoclonal antibody avidity

The ELISA method of introducing according to Gosling is carried out the mensuration of affinity of antibody, and the size of its avidity represents that with the size of affinity costant Ka formula is:

$\frac{V}{\mathrm{\&alpha;}}=K_a(1-V)$

The unit of affinity costant is the inverse of concentration, that is: molconcentration (L/moL), and the high more expression antigen-antibody bonded tightness degree of its value is high more.Its testing process is as follows:

The first step, determine antigen, antibody the best use of concentration:

1, artificial antigen is carried out 13000,26000,52000,104000 respectively, each wraps by 4 after 208000,416000 times of dilutions, 100L/ hole, 37 ℃, incubation 2h, washing, sealing;

2, antibody is carried out 7500,15000,30000,60000,120000,240000,480000 times of dilutions are added on respectively in 2,100L/ hole, room temperature incubation 1h;

3, the antibody in these two is moved in the second room temperature incubation 1h respectively;

4, these two washings are added ELIAS secondary antibody, continue to be ELISA, measure OD value A1 at last;

5, press above-mentioned steps for back two, measure OD value A2 at last.

6, according to formula $f=\frac{A_1\left(c\right)-A_2\left(c\right)}{A_1\left(c\right)}$

Calculate f value, choose all f values all less than 10% antigen, antibody dilution is 208000 times of dilutions according to the big or small definite best antigen concentration of OD value, and optimum antibody concentration is 120000 times of dilutions.

Second step, mensuration avidity

1, a series of concentration antigens of preparation are 6;

2, add equivalent series concentration antigen in being added with the EP pipe of optimum concn antibody, totally 7 manage, last pipe adds antigenic dilution, spends the night under the room temperature;

3, antigen is carried out wrapping quilt after 208000 times of dilutions simultaneously, spend the night under the room temperature, wash plate, sealing;

4, the reaction product in second step is got 100 μ L and add in the above plate hole that seals, carry out ELISA under the room temperature, measure OD value A at last;

5, according to the Scatchard formula $\frac{V}{\mathrm{\&alpha;}}=K_a(1-V)$ Calculate its slope value,

Wherein, α is the concentration of free antigen, and V is the binding antibody site and the ratio of total antibody sites, and the size of gained avidity is affinity costant Ka, is the negative of slope value.The result who obtains is that the affinity costant of monoclonal antibody 8E6 ' is 1.8*10 ¹⁰, the avidity of monoclonal antibody 6A4 ' is 1.2*10 ¹⁰

Embodiment two

Medicine cross reaction test

Undertaken by the monoclonal antibody screening indirect competitive ELISA method of setting up among the embodiment one, the monoclonal antibody of Enrofloxacin and Enrofloxacin haptens analog example hydrochloric acid Ciprofloxacin, Ofloxacine USP 23, Danofloxacin mesylate, Pefloxacin methanesulfonate, Abbott 56619, the test that is at war with of norfloxicin, lomefloxacin is diluted to different concns with these standard substance and carries out indirect competitive ELISA, draw and suppress curve, calculate the IC of competition thing ₅₀Value and cross reacting rate, its highest cross reacting rate is 1.464%.The monoclonal antibody of the Enrofloxacin of reporting in the document and the cross reacting rate of Ciprofloxacin reach 110.874, reach 71.05% with the cross reacting rate of sarafloxacin, the crossing-over rate that reacts with other quinolones also reaches more than 20%, and the monoclonal antibody of the prepared anti-Enrofloxacin of the present invention is only reacted with Enrofloxacin, with the crossing-over rate of other quinolones reaction below 1.5%, therefore, the monoclonal antibody of prepared anti-Enrofloxacin has higher specificity among the present invention, and concrete outcome sees Table 6.

Kind	％CR	Kind	％CR
				Enrofloxacin
	100％	Ciprofloxacin HCl	1.464％
				Methoxy sulfonic acid Pefloxacin	0.152％	Abbott 56619	Less than 0.01%
Ofloxacine USP 23	0.079％	Lomefloxacin	0.166％
				Norfloxicin	0.103％	Methoxy sulfonic acid danofloxacin	0.03％

The cross reaction of table 6 monoclonal antibody 8E6 ' and 6A4 ' and other quinolones

Embodiment three

Present embodiment is that monoclonal antibody 8E6 ' can be used for chicken, fishery products, the detection of enrofloxacin residual in milk and the honey setting up the applicating example that detects enrofloxacin residual ELISA method among the present invention.With chicken (market purchase) is example, illustrates that the key step that detects enrofloxacin residual in the chicken is as follows:

1, sample pre-treatments: take by weighing the 15g chicken meat sample and carry out homogenate, take by weighing the 0.5g homogenate, add the methanol solution of 1.5ml 80% with the sample diluting liquid dilution, concussion mixing 30min, 2000g, centrifugal 10min, get 100 μ L supernatants and 900 μ L sample diluting liquid mixings, get 50 μ L and detect.

2, the detection principle of test kit is the indirect competitive ELISA method among the present invention, coating antigen OVA-ENR is wrapped by on microwell plate, the Enrofloxacin standard substance or the sample that add serial dilution, the monoclonal antibody (monoclonal antibody of choosing in the present embodiment is 8E6 ') that adds anti-Enrofloxacin again, Enrofloxacin residual in the sample combines with the antibody competition of anti-Enrofloxacin with the antigen of bag quilt simultaneously, add ELIAS secondary antibody then, the TMB colour developing, OD is read in the colour developing back on microplate reader ₄₅₀, contents of enrofloxacin and sample absorbance are negative correlation in the sample, compare with typical curve, can draw corresponding residue contents of enrofloxacin.

3, the high specific of the monoclonal antibody of anti-Enrofloxacin among the present invention makes and compares with existing sample treatment, has simplified the pre-treatment step of muscle samples greatly, only directly carries out 10 times of dilutions behind needs 80% methanol extraction and promptly can be used for detecting.Because the high-affinity of antibody makes the sensitivity that detects improve greatly, standard curve range is 0.313-10ng/ml.

Adopt above method, 20 parts of chicken meat samples are detected, see Table 7, the result shows that the ELISA method of utilizing Enrofloxacin antibody to set up detects the residual of Enrofloxacin in the chicken tissue, and the coincidence rate as a result that result and high performance liquid chromatography record reaches 100%.

The sample sequence number

ELISA method detected result (ng/ml)

High performance liquid chromatography detected result (ng/ml)

1	2.047	＜30
			2	1.471	＜30
3	27.238	45.9
			4	161.3	126.8
5	175.509	167.4
			6	73.77	63.3
7	80.951	67.9
			8	2.093	＜30
9	93.75	75.4
			10	241.155	209.8
11	1.129	＜30
			12	117.002	93.1
13	206.248	223.3
			14	2.375	＜30
15	2.342	＜30
			16	0.417	＜30
17	0.972	＜30
			18	1.864	＜30
19	1.011	＜30
			20	294.737	287.4

Table 7 ELISA method and high performance liquid chromatography detected result are relatively

Embodiment four

Present embodiment is the applicating example of monoclonal antibody 6A4 ' in the colloidal gold strip of preparation enrofloxacin residual among the present invention, mainly is the enrofloxacin residual that is applied to detect in muscle (chicken, the flesh of fish, pork), milk, the honey.

Reaction principle adopts competition law that the small-molecule drug Enrofloxacin is carried out half-quantitative detection, the Enrofloxacin molecule that exists in the sample is combining along moving past the antibody protein of Cheng Zhongxian with the gold grain mark on the test strip, the coating antigen and the Enrofloxacin that are fixed on the NC film are competed binding antibody simultaneously, residual contents of enrofloxacin is inversely proportional in the colour developing power of T line and the sample, if do not have enrofloxacin residual in the sample, then golden labeling antibody all reacts with coating antigen, the T line colour developing of test strip.Represent feminine gender as C, when the T line all develops the color, when C line colour developing T line does not develop the color, then be expressed as the positive, when the C line does not develop the color, T line colour developing or do not develop the color and represent that all test strip lost efficacy.

(1) concrete operations step is as follows:

1, sample pre-treatments: the treatment process of various samples is with the sample-pretreating method in the ELISA method in present method;

2, test strip is put on the clean smooth table top, draws testing sample solution, drip 1～2 on sample pad with dropper;

3, wait for the appearance of red-purple band, read test result in the time of standing and reacting 5-10 minute, judgement later in 10 minutes is invalid.

(2) determining of test strip detectability:

1, gets test strip and lie against on the testing table, respectively with containing different concns (0,1,2,3,4,5,6,7,8,9,10ng/ml) the PBS solution of Enrofloxacin is as analyte sample fluid, drip on sample pad, reaction back 5-10min result of determination determines that the detection of test strip is limited to 2ng/ml, detectability to milk and various muscle can require to carry out different dilutions according to detecting, and 2 multiply by the detectability that different extension rates is various samples.Be exemplified below: if sample carries out 10 times of dilutions, then the detection to this sample is limited to 20ng/ml, and content is judged to be the positive greater than the sample of 20ng/ml, and content is judged to be feminine gender less than the sample of 20ng/ml.

Buy the Enrofloxacin interpolation test of carrying out different concns in the milk sample that does not contain Enrofloxacin (confirming not conform to Enrofloxacin) in market to 20 parts as stated above through high performance liquid chromatography, adopt colloid gold test paper method and high performance liquid chromatography to compare then, the result shows that the coincidence rate of two kinds of methods reaches 100%, and detected result sees Table 8.

The sample sequence number	Radioactive colloidal gold method detected result	High performance liquid chromatography detected result (ng/ml)
			1	Positive (C line colour developing T line does not develop the color)	40.8
2	Negative (C, T line all develop the color)	＜25
			3	Negative (C, T line all develop the color)	＜25
4	Negative (C, T line all develop the color)	＜25
			5	Negative (C, T line all develop the color)	＜25

6	Positive (C line colour developing T line does not develop the color)	51.7
			7	Positive (C line colour developing T line does not develop the color)	87
8	Negative (C, T line all develop the color)	＜25
			9	Negative (C, T line all develop the color)	＜25
10	Negative (C, T line all develop the color)	＜25
			11	Negative (C, T line all develop the color)	＜25
12	Positive (C line colour developing T line does not develop the color)	214.3
			13	Positive (C line colour developing T line does not develop the color)	98.9
14	Positive (C line colour developing T line does not develop the color)	49.7
			15	Positive (C line colour developing T line does not develop the color)	50.6
16	Positive (C line colour developing T line does not develop the color)	77
			17	Positive (C line colour developing T line does not develop the color)	146.8
18	Positive (C line colour developing T line does not develop the color)	43.6
			19	Negative (C, T line all develop the color)	＜25
20	Negative (C, T line all develop the color)	＜25

Table 8 Radioactive colloidal gold method and high performance liquid chromatography detected result are relatively

In addition to the implementation, the present invention can also have other embodiments.All employings are equal to the technical scheme of replacement or equivalent transformation formation, all drop on the protection domain of requirement of the present invention.

Reference:

1、Holtzapple?C?K，Buckley?S?A，Stanker?L?H.Production?andcharacterization?of?monoclonal?antibodies?against?sarafloxacin?andcros-reactivity?studies?of?related?fluoroquinolones.Journal?ofAgricultural?and?Food?Chemistry，1997，45：1984-1990.

2, Yang Liguo, " enzyme immunoassay technique ", press of Nanjing University, 1998.

Claims (3)

1. produce the hybridoma cell strain 8E6 of enrofloxacin monoclonal antibody, 8E6 is that mouse hybridoma cell is CGMCC No.3017.

2. by the described hybridoma cell strain 8E6 of claim 1 excretory monoclonal antibody 8E6 '.

3. the application of the described monoclonal antibody 8E6 ' of claim 2 in preparation detection Enrofloxacin reagent.

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